The present invention relates to compounds for use as one or more of an antiproliferative agent, a chemotherapeutic agent, an adjuvant, and antiviral agent and a cell sensitising agent. Preferably the compounds are inhibitors of protein translation.
The disruption of one or more steps in the control of protein synthesis has been associated with alterations in the cell cycle and/or regulation of cell growth. Evidence supports the concept that some translation factors are proto-oncogenes and proteins involved in translation pathways can act as key regulators of malignant progression (Hershey et al, 2000 Translational Control and Cancer, Cold Spring Harbor Laboratory Press, Cold Spring Harbor). Cancer cells generally show higher rates of protein synthesis compared to normal cells. Accordingly, deregulation of protein synthesis is emerging as a major contributor to cancer progression. Over expression of certain translation factors can lead to malignant transformation and many of the components of the translation pathways are over-expressed in cancer. A number of clinically relevant in-vivo experiments have demonstrated that inhibition of translation may be relevant for the treatment of a range of cancer types e.g. adult T-cell leukaemia, lung, breast and cervical cancer. The requirement for elevated levels of protein synthesis is a common feature of cancer cell growth; therefore it is highly likely that a wider broad spectrum of cancer types will also be amenable to treatment with this class of inhibitor.
Inhibitors of translation have shown remarkable promise for use as an adjuvant therapy in combination with chemotherapeutics such as Doxorubicin™. Rapidly proliferating tumour types such as MCF-7 breast cancer cells require relatively more protein synthesis than slower growing cancer cells such as A549 lung carcinoma cells. These slow-growing cancer cell types have relatively higher patient mortality rates five years after diagnosis due to chemoresistance to common in clinic chemotherapeutics agents such as Cisplatin™. Research has shown that cell types such as A549 lung carcinoma or SKOV3 ovarian cancer cells derive resistance to platinum based therapies through the aberrant translation of specific proteins e.g. LARP1. Experimental evidence also suggests that endogenous inhibitors of protein synthesis such as programme cell death 4 (PDCD4) modulate sensitivity to Cisplatin™ and that the levels of these endogenous inhibitors significantly correlate with disease-free survival of ovarian cancer patients.
Therapeutic modulation of protein translation by inhibition of the eIF4A RNA helicase' is a proven target for the treatment for a broad range of cancer types. Regulation of protein synthesis at the level of translation initiation (eIF4F complex containing eIF4A) is particularly important in cancer cell growth because they are metabolically highly active. This rapid growth places a heavy demand on the protein synthesis machinery. Additionally cancer cells often produce proteins that provide resistance to commonly used chemotherapeutic drugs and this resistance is determined by selective translation of key proteins i.e. dependant on eIF4A. De novo or acquired resistance to platinum chemotherapy is the leading cause of death in some cancers e.g. Ovarian, and high impact research identifies that this chemo-resistance is due to the aberrant translation of key proteins (e.g. Boussemart et al 2014, Nature, ahead of print doi:10.1038/nature13572; Wolf et al. 2014 Nature, ahead of print doi:10.1038/nature13485); also see reviews by Blagden and Willis, 2011 Nature Oncology Reviews, 8:280-291; Bitterman and Polunovsky 2012, Molecular Cancer Therapeutics, 11: 1051-1061).
The therapeutic modulation of mRNA translation; inhibition of eIF4A is therefore an excellent and well published intervention point for the treatment of a range of different cancer types; enabling a selective treatment targeted to the biology of the cancer cell. Initiation of translation is a point of convergence for multiple aberrant signalling cascades, and represents a logical approach for targeting chemotherapy-resistant cancer cells (cancer types include but are not limited to ovarian, lung, breast, leukaemia, pancreatic, kidney).
There is now compelling evidence that aberrant control of protein synthesis is linked to the progression of a range of other conditions and illnesses. Chronic conditions such as muscle wasting, autistic spectrum disorders, Alzheimer's disease, Huntingdon's disease and Parkinson's disease all share similar patterns of deregulation of protein synthesis and a number of research studies conclude that pharmacological agents targeting the protein synthesis machinery are one potential route to treatment for such conditions. Further experimental evidence also indicates that inhibitors of translation or compounds which act to modify or alter protein synthesis present an attractive opportunity as broad acting antivirals e.g. hippuristanol has been shown to effectively disrupt the control of HIV virus translation. Indications from studies using inhibitors of protein synthesis such as hippuristanol, suggest this to be a relatively non-toxic treatment option.
Herpes simplex virus HSV-1 has been shown to stimulate eIF4E phosphorylation and eIF4F complex formation in resting primary human cells. It is also known that the VHS protein (virion host shut-off), an HSV viral endonuclease, selectively associates with eIF4A and eIF4H during the viral life cycle. In addition to degrading host mRNAs, VHS is thought to play a role in regulating the temporal pattern of viral mRNA expression, through enhancement of viral RNA translation. VHS associates with eIF4A/eIF4H and, despite its endonuclease activity, this association with eIF4A has been shown to enhance translation from viral IRES (internal ribosome entry site) elements and sequences within HSV-1 5′-UTRs (Saffran et al, 2010. J. Virol. 84, 6041-6049; Reviewed by Walsh, D. (2010). Biochem. Soc. Trans. 38, 1511-1516.).
With regard to the human immunodeficiency virus (HIV), the relative expression of the two isoforms p55 and p40 of HIV-1 Gag proteins is highly dependent on the correct functioning of the translation initiation complex. The highly structured 5′-UTR of the viral p55 gene has been shown to tightly control of expression through a requirement of the eIF4F complex, especially the RNA helicase eIF4A (de Breyne et al, 2012. FEBS J. 279, 3098-3111). Additional research performed using the known inhibitor of eIF4A hippuristanol has evaluated the requirement for eIF4A in the correct translation of HIV proteins. Increasing amounts of hippuristanol inhibits the translation of the three Gag isoforms in a similar dose-response manner thus confirming a functional requirement for eIF4A in the HIV life-cycle (Locker et al, 2010. Nucleic Acids Res. 39, 2367-2377). Recent work by Plank et al, (2014. Vol. 2, Iss. 1) confirmed that Hippuristanol treatment of HeLa cells transfected with HIV-1 leader constructs inhibited IRES activity, with IC50 values in a drug-gable range (163 to 296 nM). Taken together, these results confirm that eIF4A is important in the HIV life cycle and that eIF4A presents an attractive new therapeutic target for this virus.
Inhibitors of eIF4A have been shown to have value in the prevention of influenza viral replication (e.g. WO 2013152299 A2) Recent research has demonstrated the functional impairment of eIF4A correlates with inhibition of influenza virus mRNA translation and protein synthesis, and that this helicase is essential for viral translation (data obtained from both in in vivo and in vitro analysis) (Yángüez et al, 2011. Virology. 413, 93-102). Viral mRNAs have been shown not to contain cis-acting signals that may mediate eIF4A independent translation and it is also known that trans-acting viral proteins cannot replace the function of mammalian eIF4A. Therefore inhibition of eIF4A is an attractive target to prevent the propagation and replication of the influenza virus in infected cells.
Coronaviruses (e.g. Human Coronaviruses) are recognized to cause up to a third of common colds and are also the cause of severe viral infections such as SARS. Coronavirus replication involves the generation of mRNAs with capped 5′UTRs. Coronavirus 5′UTRs e.g. those identified from SARS isolates, are relatively well conserved and the full sequence forms a complex secondary structure containing four stem-loop domains. As 5′UTR secondary structure directly correlates with the requirement for eIF4A, it is not surprising that eIF4A is considered a therapeutic target for coronavirus infection.
The translation of most of the coronaviral mRNAs is thought to be cap dependent and requires a functional translation initiation complex eukaryotic initiation factor 4F (eIF4F) (Cencic et al, 2011. J Virol. 85, 6381-6389). Inhibition of translation with the eIF4A inhibitors hippuristanol or silvestrol caused a 10- to 100-fold reduction in infectious coronavirus virus titers released from infected cells (Cencic et al, 2011. J Virol. 85, 6381-6389). This virus has been proven to be dependent on eIF4A and a significant reduction in viral progeny has been observed upon the inhibition of eIF4A (Cencic et al, 2011. J Virol. 85, 6381-6389).
Rhinoviruses are the most common viral infective agents in humans and are the major cause of the common cold. Internal ribosomal entry site elements of poliovirus (PV), human rhinovirus (HRV) and encephalomyocarditis virus (EMCV) foot-and-mouth disease virus (FMDV) groups are all inhibited by disruptive mutations to the eIF4A protein (Svitkin et al, 2001. RNA. 7, 382-394). These viruses are therefore dependant on eIF4A activity.
HCMV (human cytomegalovirus) is a herpes virus that can have serious and life threatening consequences for immunocompromised patients. As HCMV infection progresses, the abundance of core eIF4F components (eIF4A is part of the eIF4F complex) greatly increases (Walsh et al, 2005. J. Virol. 79, 8057-8064). In addition, HCMV UL69, homologous with the HSV-1 ICP27 protein, associates with eIF4A (Aoyagi et al, 2010. Proc. Natl. Acad. Sci. U.S.A. 107, 2640-2645). Pateamine A, a known inhibitor of eIF4a inhibits the replication of HCMV (see patent WO2013152299 A2). Disrupting eIF4A activity presents a therapeutic target as an antiviral for HCMV.
There is good evidence that the initiation of translation of norovirus proteins is dependent on the interaction of the VPg with the translation initiation complex (Daughenbaugh et al, 2003. EMBO J. 11, 2852-2859; Daughenbaugh et al, 2006. Virol J. 23, 3-33). Panteamine A, a proven inhibitor of eIF4A, has the potential to interfere with VPg/eIF4F complex, since it disrupts the helicase/NTPase activity of eIF4A, dysregulating its function within the eIF4F complex (Bordeleau et al, 2006. Chem Biol. 13, 1287-1295). Virologists suggest that inhibitors of eIF4A could therefore be exploited as antivirals for norovirus due to this dependency (See Rocha-Pereira and Nascimento, 2012 Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs, Antiviral Drugs—Aspects of Clinical Use and Recent Advances, Dr. Patrick Arbuthnot (Ed.), ISBN: 978-953-51-0256-4, InTech).
Recent high impact research into the cause of ASD has identified that dysregulation of protein synthesis in neuronal cells at the point of translation initiation is a primary driver of ASD symptoms (Gkogkas et al, 2013 Nature, 2013, 493:371-377; Santini et al, Nature, 2013, 493:411-415).
Work by the Sonenberg lab (Gkogkas et al, 2013. Nature, 493, 371-377) demonstrated a direct link between ASD and the relative translation of two neuroligins; these are proteins which mediate new connections between neuronal cells and regulate the composition of neurotransmitter receptors. This new research identifies that the ratio of the synthesis of these two proteins is selectively determined by the activity of the translation initiation complex and that dysregulation of synthesis drives or promotes the symptoms of ASD. Importantly it is the relative synthesis of neuroligin 1 (NLGN1) protein that is incorrectly regulated; therefore selective control of NLGN1 has been demonstrated to be a viable treatment option for ASD.
In the Gkogkas et al (2013. Nature, 493, 371-377) model therapeutic intervention to regulate NLGN1 is mediated via inhibition of eIF4E, a key protein in the translation initiation complex. However, the helicase eIF4A represents an additional and more selective new target for the control of NLGN1 synthesis; a target to elevate the symptoms of ASD. The eIF4A helicase functions to unwind long, complex and structured 5′UTRs; this is required before protein synthesis can begin. Inhibiting eIF4A selectively reduces the synthesis of proteins with greater 5′UTR secondary structure or longer length, while not inhibiting those with short 5′UTRs or unstructured UTRs. Treating cells with the coral derived inhibitor of eIF4A, hippuristanol, results selective inhibition determined by features present within the 5′UTR (e.g. Bottley et at, 2010 PLOS One, 5(9): e13030).
Although the need for chemical modifiers of translation has been well established, most current small molecule inhibitors, such as hippuristanol, are sourced from rare marine corals or sponges and prove difficult to synthesise in any meaningful quantity. Such molecules have however been successfully used to provide in-vivo evidence that this class of inhibitor is a likely successful strategy option for use in the clinic, however these molecules are source limited and as such not an available option for clinical use.